The alpha-crystallins comprise a large fraction of the soluble protein in the vertebrate lens where they were, for many years, believed to function solely as structural proteins. Lenticular alpha-crystallin is comprised of two similar subunits alphaA and alphaB, each encoded by a single gene. They are related to the small heat shock proteins, and in vitro they exhibit molecular chaperone activity, autokinase activity, and interact with, and affect the state of, several cytoskeletal components. alpha-Crystallin, especially alphaB-crystallin, has been shown to be a normal constituent of many non-lenticular tissues, and has been detected in cytoplasmic inclusion bodies found in several human pathological conditions. Toward understanding the major roles of alpha-crystallin in vivo, we previously generated alphaA- and alphaB-crystallin gene knockout mice and alphaA-/alphaB-crystallin gene double knockout mice. In collaboration with Deborah Carper's lab in the NEI, and the Children's National Medical Center Microarray Center, we are performing gene array analysis on wild type, alphaA knockout and alphaB knockout mouse lenses to define differences in gene expression patterns responsible for the cataractous phenotype in alphaA knockout mice versus the non-cataractous phenotype in alphaB knockout mice. Many of the genes consistently showing different levels of expression in the Affymetrix gene chip analysis were determined to be false positives by quantitative PCR. However, several of the identified genes have indeed been proven to be up or down regulated in the precataractous alphaA knockout lens. Further studies on the expression of these genes as a function of age and spatial distribution within the lens, are ongoing. Also, confirmation of additional differentially regulated genes, identified by Affymetrix screening, is ongoing. In collaboration with Joseph Horwitz (UCLA) we are reexamining the constituents of inclusion bodies in lenses of alphaA knockout mice. There appears to be a significant amount of gamma-crystallin in the inclusion body fraction, which increases with age, and a gradual, age-dependent loss of many crystallins in the alphaA knockout lenses, some of the changes observed in age-related cataracts. A collaboration with Usha Andley (Washington U.), has confirmed the previous observation that some lens cells cultured from alphaB knockout mice hyperproliferate and undergo ploidy changes, and has provided insights into the mechanism by which this occurs. This bolsters the hypothesis that alphaB may be essential for maintenance of normal cell cycling and genomic stability. We are currently involved in collaborations with laboratories around the world studying the functions of alphaB in the heart, muscle, nervous system, immune system, and eye.